Carbapenem-resistant Klebsiella pneumoniae among hospitalized patients in Cape Town, South Africa: molecular epidemiology and characterization

Abstract Background The molecular epidemiology of carbapenem-resistant Enterobacterales in Cape Town remains largely unknown. Objectives This study aimed to describe the molecular epidemiology, resistome, virulome and mobilome of carbapenem-resistant Klebsiella pneumoniae (CRKP) within Cape Town to guide therapy, antimicrobial stewardship and infection prevention and control practices. Methods Eighty-five CRKP isolates from hospitalized patients underwent WGS as part of a prospective, multicentre, cross-sectional study, conducted between 1 November 2020 and 30 November 2022, across public-sector and private-sector hospitals in Cape Town, South Africa. Results MLST revealed three novel types, ST6785, ST6786 and ST6787, while the most common were ST219, ST307, ST17, ST13 and ST2497. Different predominant clones were noted in each hospital. The most common carbapenemase gene was blaOXA-48-like, detected in 71% of isolates, with blaNDM detected in 5%. Notably, co-detection of two carbapenemase genes (blaOXA-48-like and blaNDM) occurred in 13% of isolates. The yersiniabactin siderophore was detected in 73% of isolates, and was most commonly associated with the ICEKp5 mobile element. All carbapenemases were located on plasmids. The genes blaOXA-181 and blaOXA-232 colocalized with a ColKP3 replicon type on assembled contigs in 83% and 100% of cases, respectively. Conclusions CRKP epidemiology in Cape Town reflects institutionally dominant, rather than regional, clones. The most prevalent carbapenemase gene was blaOXA-48-like, in keeping with CRKP epidemiology in South Africa in general. Emerging clones harbouring both blaOXA-48-like and blaNDM, such as ST17, ST2497 and the novel ST6787, are a concern due to the limited availability of appropriate antimicrobial agents in South Africa.


Introduction
The burden of difficult-to-treat MDR Gram-negative bacteria, including carbapenem-resistant Enterobacterales (CRE), is rapidly increasing in South Africa.2][3][4] While new β-lactam/β-lactamase inhibitor combinations (BLICs) are becoming more accessible, the associated cost and carbapenemase-enzyme specific activity requires local molecular epidemiology data to inform rational, cost-effective roll-out. 1he first carbapenemase enzymes detected in clinical isolates from South Africa were NDM and Klebsiella pneumoniae carbapenemase (KPC) in 2011 from the Gauteng province. 3,5Subsequently, carbapenem-hydrolysing OXA-48, and its variant OXA-181, were detected in 2012. 6More recently, a large clonal outbreak of OXA-181 in K. pneumoniae ST307 occurred across multiple provinces in the North-Eastern and North-Western regions of South Africa and a smaller outbreak of OXA-181-producing K. pneumoniae was reported from a public sector haematology/oncology unit in Cape Town. 7,8he carbapenemases IMI, 9 VIM, 3,10,11 GES, 3,11,12 KPC 3 and IMP 10,11 have been infrequently documented in South Africa, with OXA-48 and its variants, followed by NDM, currently representing the greatest proportion of carbapenemases.Further, K. pneumoniae has been the most common CPE isolated in South Africa. 3Isolates harbouring more than one carbapenemase have also been reported, with a prevalence of approximately 3.5% in CRE bloodstream infections (BSIs) among hospitalized patients surveyed under the Group for Enteric, Respiratory and Meningeal Diseases Surveillance in South Africa (GERMS-SA) of the National Institute for Communicable Diseases. 3eported data on CPEs in the Cape Town metropole have been limited in scope, consisting of institutional reports or outbreak investigations. 8,13Thus, despite frequent isolation of suspected CPEs across both the public and private sector, the molecular epidemiology and repertoire of molecular determinants of resistance remains largely unknown.A greater understanding of the CPE resistome will better inform therapeutic decisions with regard to novel antibiotics such as BLICs and repurposed older agents, while also improving antimicrobial stewardship (AMS), by facilitating evidence-based development of empirical and directed therapy protocols.Furthermore, effective infection prevention and control (IPC) interventions are hampered by limited baseline data institutionally and across the metropole.Thus, establishment of prevalent clonal types and mobile genetic elements is critical to the rapid institution of appropriate IPC measures directed at novel outbreaks.From a clinical and pathophysiological point of view, investigation of the virulome is important to guide further clinical and basic science research aimed at reducing morbidity and mortality associated with CREs.
Therefore, this study aimed to evaluate the resistome, virulome, mobilome and molecular epidemiology of the carbapenemresistant K. pneumoniae (CRKP) subset of clinical isolates from hospitalized patients enrolled in a multicentre study of CRE within the Cape Town Metropole, across all sectors.

Clinical isolates
Participants were enrolled in a prospective, multicentre, cross-sectional study between 1 November 2020 and 30 November 2022, from three public-sector hospitals [Groote Schuur Hospital (GSH), Red Cross War Memorial Children's Hospital (RCWMCH) and Tygerberg Hospital (TBH)] and three private-sector hospitals (Mediclinic Panorama, Netcare Christiaan Barnard Memorial Hospital, Netcare Blaauwberg Hospital) in Cape Town, Western Cape, South Africa.Hospitalized participants with a CRE, based on phenotypic testing, cultured during routine clinical care from any anatomical site (other than rectal swabs and stool) during the study period were eligible for inclusion.Multiple isolates from the same participant and isolates from surveillance cultures were excluded.Additional information on participant enrolment and phenotypic microbiological testing are described in the Supplementary Methods (available as Supplementary data at JAC-AMR Online).
Viable CRKP isolates (n = 85/86) underwent WGS and are included in this analysis.Non-CRKP isolates, from participants enrolled as described, did not undergo WGS.

WGS
Viable CRKP isolates (n = 85/86) were subcultured overnight on 2% blood agar in an oxygen-enriched incubator at 37°C.Genomic DNA was extracted using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research Corp., Irvine, CA, USA) followed by library preparation after DNA quality control using the Illumina DNA Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions.Library quality control was performed using an Agilent DNA 1000 kit (Agilent Technologies, CA, USA) and Qubit ™ dsDNA HS Assay Kit (Invitrogen, Life Technologies, CA, USA).Each sequencing run contained the PhiX V3 internal sequencing control (Illumina) spiked at 2%.Sequencing was performed on an Illumina ® MiSeq ™ instrument using a MiSeq ™ Reagent Kit v2 (300-cycle) (Illumina) as per the manufacturer's instructions.
In isolates where a carbapenemase-encoding gene was not detected (12%, n = 10/85), truncation of 30%-60% of the ompK35 gene was detected in five (all were ST307), one of which also had a glycine-aspartate (GD) insertion in the ompK36 extracellular loop 3 region.A further isolate (ST6785) without a detected carbapenemase-encoding gene had a significant truncation of 82% of ompK36.In general, across all isolates with and without a detected carbapenemase gene, co-detection of ompK35 truncation with ompK36 GD insertion was found in 21% (n = 18/85; all either ST307 or ST219).Truncation of ompK35 without ompK36 GD insertion was also detected in all ST2497, ST6787 and remaining, bar one, ST307 isolates.
Complete salmochelin (iro) and aerobactin (iuc) siderophore, genotoxin colibactin (clb) and regulator of mucoid phenotype (rmpADC) gene clusters were not detected in any isolates.][41] The type 1 ( fim) and type 3 (rmk) fimbriae gene clusters were complete in 87% (n = 74/85) and 93% (n = 79/85) of isolates, respectively.The kpi gene cluster, previously detected in carbapenem-resistant ST15 K. pneumoniae and associated with adherence and biofilm formation, was detected in five isolates. 42our of these isolates were ST1927 and one was ST3184.The ycfM gene, which encodes a non-fimbrial adhesin that facilitates adhesion to abiotic surfaces, was detected in all isolates. 43The LPS biosynthesis gene associated with colonization capacity, wabG, was detected in 80% (n = 68/85), while the LPS-regulating uridine diphosphate galactonate 4-epimerase gene (uge) was not detected in any isolates. 44,45The ureA and ureD genes from the urease gene cluster were detected in all isolates.
A curated set of K. pneumoniae-specific virulence genes is shown in Figure 1, along with K and O loci.All virulence genes detected using the VFDB database are shown in Table S1.

Discussion
Healthcare-onset infections continue to pose a huge medical burden to public health worldwide.K. pneumoniae, one of the WHO priority nosocomial pathogens, causes a broad spectrum of disease and frequently acquires resistance to commonly used antibiotics, particularly in ICUs. 4,46Thus, evaluating the molecular epidemiology, resistome, virulome and mobilome of CRKP isolates is pivotal to inform data-driven AMS and IPC interventions.
In this cross-sectional survey across public-sector and privatesector hospitals from Cape Town, CRKP isolates have been characterized.Similar to previous reports from South Africa, 3 bla OXA-48-like carbapenemases were the most common, with bla NDM following.Notably, co-detection of two carbapenemase enzymes (bla OXA-48-like and bla NDM ) occurred in 13% of isolates (all either ST17, ST2497 or ST6787) and was more common than detection of bla NDM alone.Emergence of MBL-producing isolates is a concern as there are no available BLICs in South Africa, at the time of writing, with in vitro activity. 47eftazidime/avibactam, in combination with aztreonam, is currently the preferred regimen for organisms with MBLs, such as NDM, and in vitro susceptibility has been shown for isolates with both MBL and serine-β-lactamases. 48,49 However, both agents have limited availability and potentially prohibitive costs in South Africa while alternative active agents, such as polymyxins and cefiderocol, have similar barriers to use and/or are associated with significant toxicity. 50,51ll carbapenemase genes were located on contigs linked to plasmids, further highlighting the need for effective IPC.Based on replicon typing of de novo-assembled plasmid sequences, all bla OXA-232 and most bla OXA-181 genes were co-localized with ColKP3 replicons.ColKP3-type plasmids carrying bla OXA-181 and bla OXA-232 have previously been described in a neonatal unit in India (ST15 and ST48) and an emergent ST15 clone in China. 47,52he IncL replicon was co-localized with bla OXA-48 on de novo-assembled plasmid sequences and its replicon was detected in isolate assemblies with bla OXA-48 that could nonetheless not be co-localized with a typeable replicon.IncL-type plasmids have previously been described as the most common plasmid type harbouring bla OXA-48 . 53hile bla NDM genes could not be localized to de novoassembled plasmid sequences, mega plasmids with replicons IncFIB(pNDM-Mar):IncHI1B(pNDM-MAR), found in ST2497 and ST6787 isolates in this study, have previously been shown to encode bla NDM . 54The replicon types IncX3, IncFIB(K), IncFII(K), IncFIA(HI1), IncM2, IncR, IncFIA, IncQ1 and IncM2 detected in other bla NDM -carrying isolates have all previously been shown to harbour bla NDM , with IncX3 being the most common. 54lthough no known carbapenemases were detected in 10 (12%) isolates, 5 had truncations of ompK35, which may contribute to carbapenem resistance, and one had a significant truncation of ompK36. 55In addition to ompK35 truncation, one of these isolates had an ompK36 GD insertion, which has been shown to cause pore constriction, resulting in elevated carbapenem MICs. 56This highlights the importance of proteome remodelling of the outer membrane in K. pneumoniae antibiotic permeability modulation. 57ron acquisition, facilitated by siderophores, is essential for K. pneumoniae replication and virulence.Unsurprisingly, the ent gene cluster was detected in all isolates.However, lipocalin 2, an innate immune protein produced by neutrophils and mucosal surfaces, specifically binds enterobactin and disrupts iron acquisition. 58Lipocalin 2 cannot bind to the siderophore yersiniabactin and thus yersiniabactin has been shown to promote respiratory tract infections. 58In this study, the ybt gene cluster was detected in approximately three-quarters of the isolates.Deletion of ybtX in two-thirds of isolates is interesting as ybtX deletion has previously been shown to be associated with reduced inflammation during lung infection with K. pneumoniae. 59urther, related to iron uptake, the kfuABC system was detected in one-third of isolates (ST13 and ST219) and has been associated with a greater tissue-invasive potential and metastatic infection. 60

Molecular epidemiology of CRKP in Cape Town
Analysis revealed several STs with significant heterogeneity between institutions.Specifically, different STs predominated in each of the respective institutions, suggesting local in-hospital circulation.The ST307 and ST17 clones, predominant in the private sector and TBH, respectively, have both been reported globally as important antimicrobial drug-resistant clones. 7,61,62he ST13 clone, predominant at RCWMCH, has previously been described as an emergent clone associated with carbapenem resistance in Europe. 63nly six isolates had a capsular locus (K2) that is associated with hypervirulence, and none of these isolates had the complete iro, iuc or rmpADC virulence gene clusters associated with the hypervirulence genotype. 64There are limited published data available on the prevalence of hypervirulent K. pneumoniae in South Africa, thus the lack of convergence of carbapenem resistance and hypervirulence, as has previously been described in China, is of uncertain significance. 65The hypervirulence-associated magA gene was also not detected in any isolates.In general, substantial heterogeneity in K locus prevalence, correlating with dominant ST, was noted between institutions with smaller diversity in O type.
This study had several limitations.Long-read sequencing was not performed, which complicated the assembly of complete plasmid sequences. 35Thus, carbapenemase gene-containing plasmidSPAdes-assembled contigs could only be replicon typed in 44% of isolates.As a result, limited inferences could be made regarding bla NDM and bla OXA-48 carbapenemase gene context, beyond detection of replicon types commonly associated with these genes in the isolate assemblies in general.A major barrier to participant recruitment was SARS-CoV-2-related policy such as changes in IPC practice.Fewer hospitals participated and participant recruitment was heavily impacted by policies such as increased approval process stringency and limitation of studyrelated staff movement within participating institutions.Additionally, travel within and outside South Africa, as well as largely temporary changes in community and hospital IPC practices, may have influenced CRKP transmission dynamics, limiting the applicability of data to current CRKP epidemiology.This siloed practice may have contributed to the study finding of institutionally dominant, rather than regional, clones.
In conclusion, this study evaluated CRKP epidemiology in Cape Town and found that the most prevalent carbapenemase gene was bla OXA-48-like , in keeping with previously described CRKP epidemiology in South Africa. 3Hypervirulent CRKP isolates were not detected in this collection.Emerging clones that may harbour both bla OXA-48-like and bla NDM , such as ST17, ST2497 and the novel ST6787, are a concern due to the limited availability of appropriate antimicrobial agents, particularly in the South African public sector.Regarding molecular epidemiology, institutionally dominant, rather than regional clones, were found.

Figure 2 .
Figure 2. Genome clustering.A neighbour-joining tree constructed from core-genome distances using GrapeTree is shown using the K. pneumoniae v3 database from the Pathogen Informatics and Modelling group at EMBL-EBI.(a) Study samples are labelled as 'Query' and database entries as 'Reference'.(b) Nodes are labelled with the ST.

Figure 4 .
Figure 4. Plasmids.(a) A bar graph of detected plasmid replicon types from assemblies, separated by institution, is shown.The bars indicate the percentage of isolates from each institution in which the plasmid replicon type was detected.The genomic context of carbapenemase genes in contigs generated by plasmidSPAdes and annotated using Prokka is shown for (b) pColKP3 with bla OXA-232 , (c) pIncL with bla OXA-48 , (d) pColKP3 with bla OXA-181 .Hypothetical proteins are not shown.